Expression of a streptococcal glucosyltransferase as a fusion to a solute-binding protein in<i>Lactobacillus fermentum</i>BR11

The aim of this study was to display a rumen bacterial β-glucanase on the cell surface of a probiotic Lactobacillus reuteri strain. The β-glucan degrading ability and the adhesion capability of the genetically modified strain were evaluated. The β-glucanase (Glu) from Fibrobacter succinogenes was fused to the C-terminus of collagen-binding protein (Cnb) from L. reuteri and then expressed by L. reuteri Pg4 as a recombinant Cnb-Glu-His(6) fusion protein. Confocal immunofluorescence microscopy and flow cytometric analysis of the transformed strain L. reuteri pNZ-cnb/glu demonstrated that Cnb-Glu-His(6) fusion protein was displayed on its cell surface. In addition, L. reuteri pNZ-cnb/glu acquired the capacity to break down barley β-glucan and showed higher adhesion capability, in comparison with the parental strain L. reuteri Pg4. To the best of the authors' knowledge, this is the first report of successful display of fibrolytic enzymes on the cell surface of intestinal lactobacilli.

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Display of Fibrobacter succinogenes β-Glucanase on the Cell Surface of Lactobacillus reuteri

Huang

Chen

Yueh

et al. 2011

J. Agric. Food Chem.

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Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from Lactobacillus fermentum BR11

Turner

Hafner

Walsh

et al. 2003

Appl Environ Microbiol

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A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His 6 ). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His 6 and CFTR peptides or His 6 peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.Over the past several years investigation into the healthpromoting, or probiotic, activity of certain strains of lactic acid bacteria in humans has been the subject of substantial research. Of particular interest are lactobacilli, which due to their nonpathogenic and mucosal surface-colonizing properties have been developed for recombinant protein delivery because they have the potential to express proteins at specific mucosal sites. For the most part, lactobacilli have been investigated as recombinant mucosal vaccines. There have been recent developments in the engineering of lactobacilli and other colonizing lactic acid bacteria so that they secrete or surface display single-chain Fv fragments with direct therapeutic effects (4, 16).Lactobacillus secretion vectors have mainly utilized the secretion signals of proteins derived from bacteria other than lactobacilli, with examples including those of the M protein from Streptococcus pyogenes, Usp45 and PrtP from Lactococcus lactis, and protein A from Staphylococcus aureus (13,31,35). Lactobacillus secretion signals that have been used for extracellular heterologous protein production include the S-layer secretion signal from Lactobacillus brevis (33) and the ␣-amylase protein from Lactobacillus casei (16,17). Protein surface display systems using lactobacilli can be divided into two classes, covalent and noncovalent anchoring systems. The covalent anchor system uses proteins that contain LP...

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Identification and Characterization of the Novel LysM Domain-Containing Surface Protein Sep from Lactobacillus fermentum BR11 and Its Use as a Peptide Fusion Partner in Lactobacillus and Lactococcus

Turner

Hafner

Walsh

et al. 2004

Appl Environ Microbiol

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Examination of supernatant fractions from broth cultures ofLactobacilli have been engineered to express a variety of heterologous proteins of interest, including antigens, enzymes, and more recently therapeutic single-chain antibodies and pathogen receptors (5,8,12,30). Certain features of lactobacilli make them attractive as recombinant protein delivery vehicles. These include their generally regarded as safe (GRAS) status; their presence in dairy, meat, and vegetable food fermentation processes; and their ability to reside naturally and persist on mucosal surfaces where they can exert certain beneficial effects to the host.A number of protein expression and targeting systems in lactobacilli have been developed which allow accumulation of the protein either intracellularly, anchored to the cell surface, or in the extracellular environment. Protein secretion and surface anchoring systems rely on the utilization of functional elements of extracellular proteins to efficiently secrete and/or anchor the heterologous protein to the cell surface. The majority of extracellular proteins in gram-positive bacteria are at some stage anchored to the cell wall either via covalent or noncovalent cell wall binding domains. Covalent attachment to the cell wall or cytoplasmic membrane occurs via the carboxyterminal LPXTG-type or amino-terminal LXXC sorting signals, respectively (18). Noncovalent attachment to the cell wall or cell wall components occurs either via specific repetitive LysM, YG, or choline-binding GW or S-layer homology domains (3, 7, 18) or via nonspecific cationic domains (2, 31). Varying amounts of both covalent and noncovalent surface anchored proteins may be released into the environment due to cell wall turnover, cell lysis, or proteolytic events (4,21,23,24).Our research is geared towards the development of heterologous protein expression systems using the targeting regions of native lactobacilli surface proteins. Our model organism is Lactobacillus fermentum BR11, which is a vaginal tract isolate of a guinea pig that has been shown to be amenable to genetic manipulation (25). Members of the L. fermentum-Lactobacillus reuteri group are commonly found inhabitants of mucosal surfaces of mammals and are one of the predominant Lactobacillus species found in the intestines of humans. To date, we have characterized a number of different surface proteins from L. fermentum BR11 and tested them as heterologous protein fusion partners. These include the noncovalently anchored cystine binding surface protein, BspA, and the covalently anchored LPXTG-containing proteins Mlp and Rlp (10,29,30,32). Here we report the identification and characterization of a novel abundant small exported protein (Sep) from L. fermen-

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Expression of a streptococcal glucosyltransferase as a fusion to a solute-binding protein inLactobacillus fermentumBR11

Cited by 4 publications

References 19 publications

Display of Fibrobacter succinogenes β-Glucanase on the Cell Surface of Lactobacillus reuteri

Display of Fibrobacter succinogenes β-Glucanase on the Cell Surface of Lactobacillus reuteri

Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from Lactobacillus fermentum BR11

Identification and Characterization of the Novel LysM Domain-Containing Surface Protein Sep from Lactobacillus fermentum BR11 and Its Use as a Peptide Fusion Partner in Lactobacillus and Lactococcus

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